The long-term scientific objectives of this application are to pursue an area of research that moves beyond a traditional structural and biochemical analysis of proteins to a more quantitative understanding of the energetics that govern protein function. Electric fields are an integral component of the energy landscapes that govern protein structure and function but very few experimental measurements of electric fields in proteins have been made. A spectroscopic technique called VSE Spectroscopy, developed by Steve Boxer's research group, will be used to perform a systematic characterization of the electric fields in two biological systems. Specific aim 1 involves comparing the electric fields in aldose and aldehyde reductase, two enzymes implicated in diabetes. This will reveal how the active sites of these closely related enzymes are electrostatically tuned to different functions, and how these differences could be used to design more potent and more specific inhibitors of aldose reductase. Specific Aim 2 involves studying the small GTPase Ras, which is mutated in a large number of human cancers. Since Ras is a highly dynamic protein, this work will enhance our understanding of how electrostatics is influenced by protein dynamics, a question about which relatively little is known. VSE Spectroscopy requires the incorporation of a nitrile group into the protein of interest to serve as an experimental reporter of electric field. Two methods have been previously developed for the incorporation of nitriles into proteins and these methods will be used to prepare proteins with probes at different sites, allowing the electrostatics to be measured over large regions of the proteins of interest. Since the Boxer lab pioneered the technique of VSE Spectroscopyit will provide an ideal environment for performing these experiments. PUBLIC HEALTH RELEVANCE: Many diseases are caused by relatively subtle perturbations to specific biological processes- for instance, cancer involves the acquisition of mutations that alter the properties of certain proteins. A comprehensive understanding of the physical properties of such proteins will reveal the origin of the aberrant behavior that arises in disease and allow us to exploit these properties for therapeutic benefit. The work proposed here with aldose reductase and Ras will improve our understanding of the involvement of these proteins in disease and lead to new ways to intervene in the disease process.